Deglycosylation to obtain stable and homogeneous Pichia pastoris-expressed N-A1 domains of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A1 domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N-A1 to test this hypothesis. The N-A1 domains were expressed as soluble protein with a C-terminal hexahistidine tag (His6-tag) in the yeast Pichia pastoris. His6-tagged N-A1 was captured from the supernatant by batch purification with copper-loaded Streamline Chelating, an immobilised metal affinity chromatography (IMAC) matrix usually utilised in expanded bed techniques. Purified N-A1 was heterogeneous with a molecular weight range from 38 to 188 kDa. Deglycosylation with endoglycosidase H (Endo H) resulted in three discrete molecular weight forms of N-A1, one partially mannosylated, one fully Endo H-digested and one fully Endo H-digested but lacking the His6-tag. These were separated by concanavalin A chromatography followed by HiTrap IMAC. The procedure resulted in single-band-purity, mannose-free N-A1. The binding interaction of MFE-23 to N-A1 was analysed by surface plasmon resonance. The affinity constants retrieved were KD = 4.49 x 10(-9)M for the P. pastoris expressed, native N-A1, and 5.33 x 10(-9) M for the Endo H-treated N-A1. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from P. pastoris. This work confirms that the CEA epitope recognised by MFE-23 resides in N-A1.     

Carcinoembryonic antigen gene family members in submandibular salivary gland: demonstration of pregnancy-specific glycoproteins by cDNA cloning

We have recently shown that human submandibular salivary gland and saliva contain a number of glycoproteins belonging to the carcinoembryonic antigen (CEA) gene family. The members of the CEA family can be divided into the CEA subgroup and the pregnancy specific beta 1 glycoprotein (PSG) subgroup. The latter glycoproteins are abundant in placenta and fetal liver. Here we report that PSG's are expressed in normal adult submandibular salivary gland. Thus, cDNA cloning and sequencing gave two clones (SG5 and SG9) which coded for glycoproteins with a domain arrangement of N-A1-A2-B2-C and a third clone (SG8) which coded for a glycoprotein with a domain arrangement of N-A1-B2-C. SG5 is identical to PSG3, and SG9 to PSG1d, while SG8 most probably corresponds to PSG2. The 3' untranslated regions of the different members of the PSG subgroup contain highly homologous segments, suggesting a common evolutionary origin.     

Refolding, purification, and crystallization of apical membrane antigen 1 from Plasmodium falciparum

Extracellular domains of malaria antigens almost invariably contain disulphide linkages but lack N- and O-linked glycosylation. The best practical approach to generating recombinant extracellular Plasmodium proteins is not established and the problems encountered when using a bacterial expression/refolding approach are discussed in detail. Limited proteolysis experiments were used to identify a relatively non-flexible core region of the Plasmodium falciparum protein apical membrane antigen 1 (AMA1), and refolding/purification was used to generate two fragments of AMA1. Several chromatographically distinct AMA1 variants were identified that are presumably differentially refolded proteins. One of these AMA1 preparations proved to be crystallizable and generated two crystal forms that diffracted X-rays to 2 A resolution.     

Bacterial production and purification of SGPI-1 and SGPI-2, two peptidic serine protease inhibitors from the desert locust, Schistocerca gregaria

The last decade, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5) and 11 additional locust peptides were identified by cDNA cloning. Furthermore, the light chain of the 155-kDa heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of 6 conserved cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa3-Cys) with the locust inhibitors. So far, for most of the PLD-related peptides the biological functions remain obscure. To obtain sufficient amounts of material to perform physiological experiments, we have optimised the production of SGPI-1-2 via a bacterial (Escherichia coli) expression system. The cDNA sequences encoding these peptides were inserted in the pMAL-2pX vector, downstream of the gene encoding the maltose-binding protein (including a signal peptide). As a consequence, both peptides were expressed as fusion proteins (2-3 mg/l) and targeted to the periplasmic space. Following a one-step affinity purification, both fusion proteins were successfully cleaved by Factor Xa and after a methanol extraction, it took only one additional RP-HPLC run to purify both peptides to homogeneity. Finally, the formation of the disulphide bridges and the biological activity of the recombinant peptides were verified by mass spectrometry and a spectrophotometric protease inhibitor assay, respectively.     

人IGF-1在大肠杆菌中的可溶表达和纯化

目的:在大肠杆菌中的可溶表达和纯化人胰岛素样生长因子1(hIGF-1)。方法:根据hIGF-1的氨基酸序列和大肠杆菌密码子偏爱性,利用重叠延伸PCR的方法合成hIGF-1DNA序列,构建表达载体,在大肠杆菌OrigamiB(DE3)中与硫氧还蛋白TrxA融合表达,并通过盐析和镍柱亲合层析进行纯化。结果:SDS-PAGE分析显示,重组融合蛋白以可溶形式存在,分子量约为28kDa,占上清总蛋白的50%以上。经盐析和镍柱亲合层析进行纯化,目标蛋白纯度可达到90%左右。结论:复合干扰素在大肠杆菌中的高效可溶表达。     

Isolation and characterization of cDNA clones for Humly9: the human homologue of mouse Ly9

Ly9 is a mouse cell membrane antigen found on all lymphocytes and coded for by a gene that maps to chromosome 1. We previously described the isolation and characterization of a full-lenght cDNA clone for mouse Ly9. Using cross-species hybridization we isolated cDNA clones encoding the human homologue Humly9. Analysis of the predicted protein sequence suggests that the extra-cellular portion of the Humly9 molecules is composed of four Ig-like domains: a V domain (V) without disulphide bonds and a truncated C2 domain (tC2) with two disulphide bonds, a second V domain without disulphide bonds and a second tC2 with two disulphide bonds, i.e., as V-tC2-V-tC2. The gene encoding Humly9 was mapped to chromosome 1 by analysis of human/hamster hybrids, and more specifically to the 1q22 region by in situ hybridization. The protein sequence data support the view that Humly9 belongs to the immunoglobulin-superfamily subgroup which includes CD48, CD2, and LFA-3.The nucleotide sequence data reported in this paper has been submitted to the GenBank nucleotide sequence database has been assigned the accession number L42621     

Cellulose affinity purification of fusion proteins tagged with fungal family 1 cellulose-binding domain

N- or C-terminal fusions of red-fluorescent protein (RFP) with various fungal cellulose-binding domains (CBDs) belonging to carbohydrate binding module (CBM) family 1 were expressed in a Pichia pastoris expression system, and the resulting fusion proteins were used to examine the feasibility of large-scale affinity purification of CBD-tagged proteins on cellulose columns. We found that RFP fused with CBD from Trichoderma reesei CBHI (CBD(Tr)(CBHI)) was expressed at up to 1.2g/l in the culture filtrate, which could be directly injected into the cellulose column. The fusion protein was tightly adsorbed on the cellulose column in the presence of a sufficient amount of ammonium sulfate and was efficiently eluted with pure water. Bovine serum albumin (BSA) was not captured under these conditions, whereas both BSA and the fusion protein were adsorbed on a phenyl column, indicating that the cellulose column can be used for the purification of not only hydrophilic proteins but also for hydrophobic proteins. Recovery of various fusion proteins exceeded 80%. Our results indicate that protein purification by expression of a target protein as a fusion with a fungal family 1 CBD tag in a yeast expression system, followed by affinity purification on a cellulose column, is simple, effective and easily scalable.     

Purification and characterization of a recombinant Yersinia pestis V-F1 "Reversed" fusion protein for use as a new subunit vaccine against plague

We previously developed a unique recombinant protein vaccine against plague composed of a fusion between the Fraction 1 capsular antigen (F1) and the V antigen. To determine if overall expression, solubility, and recovery of the F1-V fusion protein could be enhanced, we modified the original fusion. Standard recombinant DNA techniques were used to reverse the gene order such that the V antigen coding sequence was fused at its C-terminus to the N-terminus of F1. The F1 secretion signal sequence (F1S) was subsequently fused to the N-terminus of V. This new fusion protein, designated F1S-V-F1, was then co-expressed with the Y. pestis Caf1M periplasmic chaperone protein in BL21-Star Escherichia coli. Recombinant strains expressing F1-V, F1S-F1-V, or F1S-V-F1 were compared by cell fractionation, SDS-PAGE, Western blotting, and suspension immunolabelling. F1S-V-F1 exhibited enhanced solubility and secretion when co-expressed with Caf1M resulting in a recombinant protein that is processed in a similar manner to the native F1 protein. Purification of F1S-V-F1 was accomplished by anion-exchange and hydrophobic interaction chromatography. The purification method produced greater than 1mg of purified soluble protein per liter of induced culture. F1S-V-F1 polymerization characteristics were comparable to the native F1. The purified F1S-V-F1 protein appeared equivalent to F1-V in its ability to be recognized by neutralizing antibodies.     

cDNA and gene analyses imply a novel structure for a rat carcinoembryonic antigen-related protein

The gene encoding the human tumor marker carcinoembryonic antigen (CEA) belongs to a gene family which can be subdivided into the CEA and the pregnancy-specific glycoprotein subgroups. The corresponding proteins are members of the immunoglobulin superfamily, characterized through the presence of one IgV-like domain and a varying number of IgC-like domains. Since the function of the CEA family is not well understood, we decided to establish an animal model in the rat to study its tissue-specific and developmental stage-dependent expression. To this end, we have screened an 18-day rat placenta cDNA library with a recently isolated fragment of a rat CEA-related gene. Two overlapping clones containing the complete coding region for a putative 709 amino acid protein (rnCGM1; Mr = 78,310) have been characterized. In contrast to all members of the human CEA family, this rat CEA-related protein consists of five IgV-like domains and only one IgC-like domain. This novel structure, which has been confirmed at the genomic level might have important functional implications. Due to the rapid evolutionary divergence of the rat and human CEA gene families it is not possible to assign rnCGM1 to its human counterpart. However, the predominant expression of the rnCGM1 gene in the placenta suggests that it could be analogous to one of the human pregnancy-specific glycoprotein genes.     

Design and production of a novel antimicrobial fusion protein in Escherichia coli

In recent years, antimicrobial peptides (AMPs) have attracted increasing attention. The microbial cells provide a simple, cost-effective platform to produce AMPs in industrial quantities. While AMP production as fusion proteins in microorganisms is commonly used, the recovery of AMPs necessitates the use of expensive proteases and extra purification steps. Here, we develop a novel fusion protein DAMP4-F-pexiganan comprising a carrier protein DAMP4 linked to the AMP, pexiganan, through a long, flexible linker. We show that this fusion protein can be purified using a non-chromatography approach and exhibits the same antimicrobial activity as the chemically synthesized pexiganan peptide without any cleavage step. Activity of the fusion protein is dependent on a long, flexible linker between the AMP and carrier domains, as well as on the expression conditions of the fusion protein, with low-temperature expression promoting better folding of the AMP domain. The production of DAMP4-F-pexiganan circumvents the time-consuming and costly steps of chromatography-based purification and enzymatic cleavages, therefore shows considerable advantages over traditional microbial production of AMPs. We expect this novel fusion protein, and the studies on the effect of linker and expression conditions on its antimicrobial activity, will broaden the rational design and production of antimicrobial products based on AMPs.

     

Development of the Fc-III Tagged Protein Expression System for Protein Purification and Detection

In the present work, we developed the Fc-III tagged protein expression system for protein purification and detection. The Fc-III sequence encodes for a 13 residue peptide and this peptide is cyclized by disulfide bond formation when the fusion protein is expressed. The Fc-III-fusion proteins selectively bind to immunoglobulin Fc domains (IgG-Fc) expressed from E. coli. We showed the efficient purification of Fc-III tagged proteins by immobilized non-native IgG-Fc and the detection of the cellular locations of fusion proteins by fluorescent-conjugated IgG-Fc. Our results prove that Fc-III tagged protein expression system is a simple and efficient tool for protein purification and detection and is a useful addition to the biochemistry and proteomics toolbox.     

A fusion‐protein approach enabling mammalian cell production of tumor targeting protein domains for therapeutic development

A single chain Fv fragment (scFv) is a fusion of the variable regions of heavy (V_H) and light (V_L) chains of immunoglobulins. They are important elements of chimeric antigen receptors for cancer therapy. We sought to produce a panel of 16 extracellular protein domains of tumor markers for use in scFv yeast library screenings. A series of vectors comprising various combinations of expression elements was made, but expression was unpredictable and more than half of the protein domains could not be produced using any of the constructs. Here we describe a novel fusion expression system based on mouse TEM7 (tumor endothelial marker 7), which could facilitate protein expression. With this approach we could produce all but one of the tumor marker domains that could not otherwise be expressed. In addition, we demonstrated that the tumor associated antigen hFZD10 produced as a fusion protein with mTEM7 could be used to enrich scFv antibodies from a yeast display library. Collectively our study demonstrates the potential of specific fusion proteins based on mTEM7 in enabling mammalian cell production of tumor targeting protein domains for therapeutic development.     

口蹄疫病毒P1基因在大肠杆菌中的高效表达及其生物活性的初步分析

将口蹄疫病毒 (FMDV)结构蛋白基因P1的完整cDNA序列插入原核表达性载体pGEX KG中 ,使P1基因与GST融合 ,获得融合表达质粒pKG P1,转化E .coliBL₂₁ (DE3) ,经IPTG诱导 ,SDS PADE结果表明GST P1融合蛋白获得高效表达 ,Western blot检测证实表达的融合蛋白具有免疫学活性 ,表达产物主要存在于细菌裂解液上清中。进一步采用GST纯化试剂盒纯化P1蛋白并作为诊断抗原 ,建立了P1 ELISA诊断方法 ,与FMD间接血凝 (IHA)检测方法平行检测 86 4份血清样品 ,总的符合率达87%。     

Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase

Prokaryotic expression of polypeptides as fusion proteins with glutathione-S-transferase has recently been reported as a one-step means of purifying recombinant protein. The usefulness of the glutathione-S-transferase/glutathioneagarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insuluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione-S-transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one-step purification utilizing this scheme requires proper folding of recombinant glutathione-S-transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione-agarose affinity purification. Low-temperature induction of fusion protein synthesis, but not incubation with anion-exchange resins, led to improved one-step purification of glutathione-S-transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity-purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione-S-transferase.     

抗癌胚抗原单链抗体基因与核心链霉亲和素基因融合在昆虫细胞中的表达

将抗癌胚抗原单链抗体基因与核心链霉亲和素基因融合插入昆虫杆状病毒供体质粒 pFastBacHTa中 ,在粉纹夜蛾Tn 5B1 4细胞中进行表达。SDS PAGE分析结果表明 ,表达产物分子量为 4 1kD左右 ,Western印迹分析结果表明 ,以HRP标记的生物素进行蛋白质印迹在 4 1kD处可见表达条带 ,表明融合蛋白能特异性的与生物素结合 ,放射免疫分析表明重组杆状病毒表达产生的ScFv CS蛋白能特异性结合癌胚抗原     

Production of bifunctional single-chain antibody-based fusion proteins in <Emphasis Type="Italic">Pichia </Emphasis><Emphasis Type="Italic">pastoris</Emphasis> supernatants

Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.     

The specificity for the differentiation blocking activity of carcinoembryonic antigen resides in its glycophosphatidyl-inositol anchor

Ectopic expression of various members of the human carcinoembryonic antigen (CEA) family of intercellular adhesion molecules in murine myoblasts either blocks (CEA, CEACAM6) or allows (CEACAM1) myogenic differentiation. These surface glycoproteins form a subset of the immunoglobulin (Ig) superfamily and are very closely related, but differ in the precise sequence of their external domains and in their mode of anchorage to the cell membrane. CEA and CEACAM6 are glycophosphatidyl-inositol (GPI) anchored, whereas CEACAM1 is transmembrane (TM) anchored. Overexpression of GPI-linked neural cell adhesion molecule (NCAM) p125, also an adhesion molecule of the Ig superfamily, accelerates myogenic differentiation. The molecular requirements for the myogenic differentiation block were investigated using chimeric constructs in which the COOH-terminal hydrophobic domains of CEA, CEACAM1, and NCAM p125 were exchanged. The presence of the GPI signal sequence specifically from CEA in the chimeras was sufficient to convert both CEACAM1 and NCAM into differentiation-blocking proteins. Conversely, CEA could be converted into a neutral protein by exchanging its GPI anchor for the TM anchor of CEACAM1. Since the external domains of CEA, CEACAM1, and NCAM can all undergo homophilic interactions, and mutations in the self-adhesive domains of CEA abrogate its differentiation-blocking activity, the structural requirements for differentiation-inhibition are any self-adhesive domains attached to the specific GPI anchor derived from CEA. We therefore suggest that biologically significant functional information resides in the processed extreme COOH terminus of CEA and in the GPI anchor that it determines.     

Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype

The new antigen receptor (IgNAR) antibodies from sharks are disulphide bonded dimers of two protein chains, each containing one variable and five constant domains. Three types of IgNAR variable domains have been discovered, with Type 3 appearing early in shark development and being overtaken by the antigen-driven affinity-matured Type 1 and 2 response. Here, we have determined the first structure of a naturally occurring Type 2 IgNAR variable domain, and identified the disulphide bond that links and stabilizes the CDR1 and CDR3 loops. This disulphide bridge locks the CDR3 loop in an "upright" conformation in contrast to other shark antibody structures, where a more lateral configuration is observed. Further, we sought to model the Type 3 isotype based on the crystallographic structure reported here. This modeling indicates (1) that internal Type 3-specific residues combine to pack into a compact immunoglobulin core that supports the CDR loop regions, and (2) that despite apparent low-sequence variability, there is sufficient plasticity in the CDR3 loop to form a conformationally diverse antigen-binding surface.